The long-range goal of this research is to exploit the multilineage differentiation capacity of bone marrow stem cells in the development of novel cellular therapies. This application builds on the demonstration that clinical transplantation of whole bone marrow (BMT) results in donor cells engraftment in the bones of patients with osteogenesis imperfecta, a genetic bone disorder. Subsequent transplantation of isolated gene-marked mesenchymal stem cells also led to donor cell engraftment in bone but at a much lower level that seen with BMT, suggesting that the major source of osteogenic activity in bone marrow is not represented in the plastic-adherent population of marrow stem cells. Thus, a murine BMT model was transplanted with retroviraly-transduced donor cells in an effort to identify the putative stem cell responsible for the unexpected effect of BMT on osteogenesis. In addition to blood, the plastic-nonadherent marrow cells gave rise to 20-50% of the osteoblasts identified in bone. Clonal analysis by retroviral integration site studies revealed that a single nonadherent marrow cell had given rise to both blood and bone cells, indicating the existence of a multipotent marrow with both hematopoietic and osteopoietic differentiation capacity. Aim 1 seeks to define the phenotype and developmental potential of this cell, using GFP-expressing transgenic mice as donors of candidate marrow cell subsets whose properties will be tested and compared in a murine transplantation model. The multilineage differentiation capacity of the strongest candidates will be verified by rigorous clonal analysis (comparison of retroviral integration sites) after single-cell transplantation. Related experiments will determine the requirement for accessory cells in the commitment of stem cells to differentiate in the osteocytic pathway. In Aim 2, the goal is to test our working hypothesis in a rhesus macaque monkey model, investigating the capacity of gene-marked CD34+ and CD34- (SP) nonadherent marrow cells to clonally regenerate the hematopoietic and osteopoietic compartments. Results of this aim will provide valuable estimates of the feasibility of marrow stem cell-based therapy for human bone diseases or injury. Finally, in Aim 3, efforts will be made to identify growth factors that might stimulate the differentiation of marrow stem cells toward the osteocytic lineage, thus addressing the general issue of low-level engraftment by tissue (adult) stem cells. Collectively, the data generated by this 5-year project will help to clarify several outstanding questions in stem cell biology and the therapeutic relevance of marrow stem cells with apparent multilineage potential.